Endothelial cells are specialized cells which form the lining of the heart and the blood vessels. Because of their direct contact with the circulating blood, a number of proposals have been made to genetically engineer these cells and use them as "in vivo" drug delivery systems. See, for example, Culliton, B. J. 1989. "Designing Cells to Deliver Drugs," Science. 246:746-751; and Zwiebel, J. A., S. M. Freeman, P. W. Kantoff, K. Cornetta, U. S. Ryan, and W. F. Anderson. 1989. "High-Level Recombinant Gene Expression in Rabbit Endothelial Cells Transduced by Retroviral Vectors," Science. 243:220-222 (transfer of a human adenosine deaminase gene and a rat growth hormone gene to aortic endothelial cells using a retroviral vector and demonstration of the secretion of rat growth hormone from such cells after seeding onto a synthetic vascular graft).
Endothelial cells are known to play an important role in the pathogenesis of atherosclerotic plaques, as well as in the success or failure of various surgical procedures, including vascular stent implantation, coronary angioplasty, and coronary bypass surgery using autologous veins or arteries or synthetic materials, such as, dacron or expanded polytetrafluorethylene.
Endothelial cells affect both the disease process and efforts to reconstruct damaged vessels because, among other things, they can: 1) alter the thrombogenic properties of the blood vessel wall, 2) modulate smooth muscle cell proliferation and migration, and 3) affect vascular smooth muscle tone through multiple pathways including the renin-angiotensin system (i.e., the system wherein the proteolytic enzyme renin cleaves two amino acids from angiotensin I to produce the hypertensive agent angiotensin II).
With regard to their interaction with the renin-angiotensin system, investigators have demonstrated in vitro that many of the constituents of that system, including angiotensinogen, renin, angiotensin-converting enzyme, and angiotensin II receptors, are contained within endothelial cells thus forming an autocrine angiotensin system. See Lilly, L. S., R. E. Pratt, R. W. Alexander, D. M. Larson, K. E. Ellison, M. A. Gimbrone, and V. J. Dzau. 1985. "Renin expression by vascular endothelial cells in culture," Circ. Res. 57:312-318; Caldwell, P. R. B., B. C. Seegal, and K. C. Hsu. 1976. "Angiotensin-converting enzyme: vascular endothelial localization," Science (Wash. D.C.). 191:1050-1051; Ryan, U. S., J. W. Ryan, C. Whitaker, and A. Chiu. 1976. "Localization of angiotensin converting enzyme (kininase II). II. Immunocytochemistry and immunofluorescence," Tissue Cell. 8:125-145; Johnson, A. R., and E. G. Erdos. 1977. "Metabolism of vasoactive peptides by human endothelial cells in culture: angiotensin I converting enzyme (kininase II) and angiotensinase," J. Clin. Invest. 59:684-695; and Patel, J. M., F. R. Yarid, E. R. Block, and M. K. Raizda. 1989. "Angiotensin receptors in pulmonary arterial and aortic endothelial cells," Am. J. Physiol 256:C987-C993. Also, interruption of the endothelial autocrine angiotensin system, with either the angiotensin-converting enzyme inhibitor lisinopril or the angiotensin II receptor antagonist sar.sup.1, ile.sup.8 -angiotensin II, has been shown to lead to increased endothelial cell migration and urokinase-like plasminogen activator (u-PA) activity. See Bell, L. and J. A. Madri. 1990. "Influence of the angiotensin system on endothelial and smooth muscle cell migration," Am. J. Pathol. 137:7-12.
In terms of clinical practice, restenosis following coronary angioplasty comprises a significant medical problem since it occurs within six months following 30-50% of the procedures performed and is associated with substantial patient morbidity and health care expenditures. All angioplasties cause removal of the endothelial cell lining of the blood vessel. The principal reasons for the restenosis are acute thrombus formation due to loss of the anti-thrombotic surface provided by the endothelial cells and neointima formation due to unchecked smooth muscle cell stimulation by blood-borne cells, again due to the loss of the protective endothelial cell layer.
For example, Fishman, J. A., G. B. Ryan, M. J. Karnovsky. 1975. "Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening," Laboratory Investigation. 32:339-351 show that loss of endothelial cells with denudation injury to the blood vessel wall is correlated with the subsequent formation of a neointima, or ingrowth of smooth muscle cells from the media into the intima and elaboration of increased amount of extracellular matrix material resulting in a new intima. Schwartz, S. M., C. C. Haudenschild, and E. M. Eddy. 1978. "Endothelial regeneration: I. Quantitative analysis of initial stages of endothelial regeneration in rat aortic intima," Laboratory Investigation. 38:568-580 show that following denudation injury to an artery, in vivo, as would be expected following angioplasty or saphenous vein graft harvesting, remaining endothelial cells migrate to restore luminal integrity, and further Haudenschild, C. C. and S. M. Schwartz. 1979. "Endothelial regeneration: II. Restitution of endothelial continuity," Laboratory Investigation. 41:407-418 show that injured vessel areas which are rapidly covered by a continuous layer of endothelium are protected from the development of neointima formation, or vessel lumen occlusion. Reidy, M. A. and S. M. Schwartz. 1981. "Endothelial regeneration: III. Time course of intimal changes after small defined injury to rat aortic endothelium," Laboratory Investigation. 44:301-308 also show that rapid coverage of the injured area is beneficial since removal of only a small number of endothelial cells from the vessel lumen allows rapid recoverage of the area with endothelial cells and prevents the development of neointima formation, or vessel lumen occlusion. Further, Madri, J. A., M. A. Reidy, 0. Kocher, and L. Bell. 1989. "Endothelial cell behavior following denudation injury is modulated by TGF-B1 and fibronectin," Laboratory Investigation. 60:755-765 show that changes in in vivo endothelial cell migration correlate with in vitro endothelial cell migration assays. Hence, rapid coverage of a denuded vessel segment, after angioplasty or following saphenous vein harvesting for bypass surgery for example, is an important parameter in preventing the vessel occlusion that commonly follows these procedures.
Occlusion of peripheral arterial and coronary artery bypass grafts is a further frequent and important clinical finding. Two-thirds of the saphenous vein coronary bypass grafts are either severely diseased or entirely occluded by six to eleven years following bypass surgery. Peripheral arterial bypass grafts have a similar fate. The occlusion is due to loss of endothelial cells from the surface of the vein graft during harvesting of the graft and at the time of initial surgery.
Synthetic grafts also exhibit high rates of occlusion. Initially, grafts of this type are not endothelialized. This results in a substantial incidence of early occlusion due to thrombosis. With time, the grafts become partially re-endothelialized by migration of arterial endothelial cells from the proximal and distal anastomotic sites or from ingrowth of capillary endothelial cells through the porous synthetic graft onto the luminal surface. However, the process of endothelial cell migration is normally slow and does not permit total coverage of the graft by arterial endothelial cells. Further, ingrowing capillary endothelial cells are less capable of inhibiting clot formation than arterial endothelial cells. Attempts to reseed peripheral grafts with autologous endothelial cells have demonstrated that incomplete coverage of the graft at the time of seeding results in graft closure and lack of clinical benefit of the seeding procedure.
Thus, Zilla, P., R. Fasol, M. Deutsch, T. Fischlein, E. Minar, A. Hammerle, 0. Krapicka, and M. Kadietz. 1987. "Endothelial cell seeding of polytetrafluoroethylene vascular grafts in humans: A preliminary report," Journal of Vascular Surgery. 6:535-541 and Fasol, R., P. Zilla, M. Deutsch, M. Grimm, T. Fischlein, and G. Laugfer. 1989. "Human endothelial cell seeding: Evaluation of its effectiveness by platelet parameters after one year," Journal of Vascular Surgery. 9:432-436 describe the absence of any significant improvement in platelet factors or function, platelet uptake on the graft surface, or distal blood flow up to one year after peripheral arterial bypass with a synthetic graft in patients who received synthetic grafts only partially coated with autologous endothelial cells. Ortenwall, P., H. Wadevik, J. Kutti, and B. Risberg. 1990. "Endothelial cell seeding reduces thrombogenicity of Dacron grafts in humans," Journal of Vascular Surgery. 11:403-410 did not observe any significant improvement in graft patency in patients who received synthetic graft partially coated with autologous endothelial cells. Thus reseeding of synthetic grafts, or autologous grafts or denuded angioplasty sites, with endothelial cells will not result in clinical therapeutic benefit unless there is virtually complete coverage of the vessel segment with a continuous layer of endothelium.
Genetic engineering of endothelial cells has been performed by a number of workers in the art. For example, Nabel, E. G., G. Plautz, F. M. Boyce, J. C. Stanley, and G. J. Nabel. 1989. "Recombinant Gene Expression in Vivo Within Endothelial Cells of the Arterial Wall," Science. 244:1342-1343, describe experiments in which a gene for the marker protein .beta.-galactosidase was transferred to endothelial cells using a retroviral vector and the thus modified cells were seeded onto the walls of an artery in vivo using a double balloon catheter to isolate the section of the artery where the seeding took place. Nabel et al. report that up to four weeks after surgery, the seeded arteries were found to contain endothelial cells which expressed .beta.-galactosidase.
Wilson, J. M., L. K. Birinyi, R. N. Salomon, P. Libby, A. D. Callow, and R. C. Mulligan. 1989. "Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells," Science. 244:1344-1346, describe similar work wherein a .beta.-galactosidase gene was transferred to endothelial cells using a retrovirus, the modified cells were seeded onto synthetic grafts, and the grafts were implanted in the carotid arteries of dogs. Five weeks later, the grafts were removed and found to still contain the genetically modified endothelial cells along their luminal surfaces.
Along these same lines, Dichek, D. A., R. F. Neville, J. A. Zwiebel, S. Freeman, M. B. Leon, and W. F. Anderson. 1989. "Seeding of Intravascular Stents with Genetically Engineered Endothelial Cells," Circulation. 80:1347-1353, describe the seeding of stainless steel stents with genetically engineered endothelial cells carrying in some cases a .beta.-galactosidase gene and in others a human tissue-type plasminogen activator (TPA) gene. See also PCT Patent Publication No. WO 90/06997 (transfer of .beta.-galactosidase, rat growth hormone, and human adenosine deaminase, CD-4, and TPA genes to endothelial cells and seeding of silicon coated polyurethane grafts and stainless steel stents with genetically engineered cells); and Zweibel et al. 1989, supra.
Direct in vivo transformation of arterial endothelial cells using retroviral particles or plasmid carrying liposomes is described in Nabel, E. G., G. Plautz, and G. J. Nabel. 1990. "Site-Specific Gene Expression in Vivo by Direct Gene Transfer into the Arterial Wall," Science. 249:1285-1288. .beta.-galactosidase was again used as a marker protein, and evidence of transformation could be found 21 weeks after transfection.
The cellular src gene (c-src gene) was first identified in the late 1970's. See Stehelin, D., H. E. Varmus, J. M. Bishop, and P. K. Vogt. 1976. "DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA," Nature. 260:170-173; and Spector, D., H. E. Varmus, and J. M. Bishop. 1978a. "Nucleotide sequences related to the transforming gene of avian sarcoma virus are present in DNA of uninfected vertebrates," Proc. Nat. Acad. Sci. USA. 75:4102-4106. The gene appears to be present in all animal species and is highly conserved. It encodes a 60,000 dalton protein, tyrosine kinase, which is localized on the cytoplasmic side of the plasma membrane. The c-src protein will be designated herein as pp60.sup.c-src.
pp60.sup.c-src is a representative molecule of the src-family of membrane-bound tyrosine kinases including, but not limited to yes, lck, and fyn. (See C. A. Koch, D. Anderson, M. F. Moran, C. Ellis, and T. Pawson. 1991 "SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins," Science. 252:668-674.) Certain critical and highly conserved noncatalytic domains in the src family of tyrosine kinases are called Src homology (SH) regions 2 and 3 and are involved in protein-protein interactions. These Src-homology domains are also found in a series of critical molecules, including, but not limited to fyn, lck, yes, PLC, p85, tensin, crk, vav, GAP, fps, arg, dabl, hck, blk, fgr, and nck. These domains are believed to regulate various cell effects of src and related molecules including, but not limited to, signal transduction pathways of tyrosine kinase receptors.
The sequence of the c-src gene has been known for some time. See Takeya, T. and H. Hanafusa. 1983. "Structure and Sequence of the Cellular Gene Homologous to the RSV src Gene and the Mechanism for Generating the Transforming Virus," Cell. 32:881-890. A copy of the nucleotide sequence for the coding region of this gene in the chicken and of the resulting pp60.sup.c-src protein as published by Takeya and Hanafusa appear as SEQ. ID. NOS. 1 and 2, respectively, set forth below. The corresponding human sequences are set forth as SEQ. ID. NOS. 3 and 4. See Anderson, S. K., C. P. Gibbs, A. Tanaka, H. Kung, and D. Fujita. 1985. "Human Cellular src Gene: Nucleotide Sequence and Derived Amino Acid Sequence of the Region Coding for the Carboxy-Terminal Two-Thirds of pp60.sup.c-src," Molecular and Cellular Biology. 5:1122-1129 and Tanaka, A., C. P. Gibbs, R. R. Arthur, S. K. Anderson, H. Kung, and D. Fujita. 1987. "DNA Sequence Encoding the Amino-Terminal Region of the Human c-src Protein: implications of Sequence Divergence among src-Type Kinase Oncogenes," Molecular and Cellular Biology. 7:1978-1983.
Various functions and properties of the c-src gene have been described in the literature. For example, Shalloway, D., P. M. Coussens, and P. Yaciuk. 1984. "Overexpression of the C-src Protein Does Not Induce Transformation of NIH 3T3 Cells," Proc. Natl. Acad. Sci. USA. 81:7071-7075, have shown that genetically engineered mouse NIH 3T3 fibroblast cells which overexpress pp60.sup..cent.-sr.cent. are not malignant. Azarnia, R. S. Reddy, T. E. Kmiecik, D. Shalloway, and W. R. Loewenstein. 1988. "The Cellular src Gene Product Regulates Junctional Cell-to--Cell Communication," Science. 23:398-401, have shown that overexpression of pp60.sup.c-src in NIH 3T3 cells causes a reduction in cell-to-cell transmission of molecules in the 400 to 700 dalton range. See also Loewenstein, W. R., and R. Azarnia. 1988. "Regulation of Intercellular Communication and Growth by the Cellular src Gene," Annals New York Academy of Sciences. 551:337-346. Soriano, P., C. Montgomery, R. Geske, and A. Bradley. 1991. "Targeted Disruption of the c-src Proto-Oncogene Leads to Osteopetrosis in Mice," Cell. 64:693-702, have shown that mutation of the c-src gene results in a marked decrease in the rate of bone resorption in mice, i.e., osteopetrosis, thus suggesting that the normal c-src gene plays a role in bone formation.
In addition to the foregoing, Warren, S. L., L. M. Handel and W. J. Nelson. 1988. "Elevated expression of pp60.sup.c-src alters a selective morphogenetic property of epithelial cells in vitro without a mitogenic effect," Mol. Cell. Biol. 8:632-646, have shown that the overexpression of pp60.sup.c-src in Madin-Darby canine kidney cells causes those cells to undergo changes in shape, including the formation of elongated cell processes having lengths in the range of 100 to 200 microns.
A gene related to the c-src gene is the oncogene v-src which forms part of the genome of the Rous sarcoma virus and causes that virus to produce sarcomas in chickens. See Takeya and Hanafusa, supra; and Hunter, T. 1987. "A Tail of Two src's: Mutatis Mutandis," Cell. 49:1-4. As with many malignant cells, cells infected with the Rous sarcoma virus have been found to exhibit increased production of urokinase-type plasminogen activator (u-PA). In particular, Bell, S. M., R. W. Brackenbury, N. D. Leslie and J. L. Degen. 1990. "Plasminogen activator gene expression is induced by the src oncogene product and tumor promoters," J. Biol. Chem. 265:1333-1338, have correlated the increased production of u-PA after transformation of chicken embryo fibroblasts by the Rous sarcoma virus with an increase in cellular u-PA mRNA.
Significantly, none of the foregoing references in any way discloses or suggests the surprising results achieved by the present invention wherein increased expression of pp60.sup.c-src by genetically engineered endothelial cells has been found to result in 1) enhanced migration of the cells, i.e., an enhanced ability to repair the endothelial lining of damaged vessels and/or an enhanced ability to form an endothelial lining on grafts or stents; and 2) enhanced urokinase-type plasminogen activator activity, i.e., an enhanced ability to dissolve or prevent the formation of the thrombi normally associated with vascular surgical procedures.